Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures

Background: The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 similar to 15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A(2A)R) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures. Results: Bioreactor cultures yielded an approximately five times increase in cell density (OD600 similar to 75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A(2A)R, and therefore more suitable for further functional and structural studies. Conclusion: Large-scale expression of the A(2A)R in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.

Planning rigid body motions using elastic curves

This paper tackles the problem of computing smooth, optimal trajectories on the Euclidean group of motions SE(3). The problem is formulated as an optimal control problem where the cost function to be minimized is equal to the integral of the classical curvature squared. This problem is analogous to the elastic problem from differential geometry and thus the resulting rigid body motions will trace elastic curves. An application of the Maximum Principle to this optimal control problem shifts the emphasis to the language of symplectic geometry and to the associated Hamiltonian formalism. This results in a system of first order differential equations that yield coordinate free necessary conditions for optimality for these curves. From these necessary conditions we identify an integrable case and these particular set of curves are solved analytically. These analytic solutions provide interpolating curves between an initial given position and orientation and a desired position and orientation that would be useful in motion planning for systems such as robotic manipulators and autonomous-oriented vehicles.

Integrable quadratic Hamiltonians on the Euclidean group of motions

In this paper, we discuss the problem of globally computing sub-Riemannian curves on the Euclidean group of motions SE(3). In particular, we derive a global result for special sub-Riemannian curves whose Hamiltonian satisfies a particular condition. In this paper, sub-Riemannian curves are defined in the context of a constrained optimal control problem. The maximum principle is then applied to this problem to yield an appropriate left-invariant quadratic Hamiltonian. A number of integrable quadratic Hamiltonians are identified. We then proceed to derive convenient expressions for sub-Riemannian curves in SE(3) that correspond to particular extremal curves. These equations are then used to compute sub-Riemannian curves that could potentially be used for motion planning of underwater vehicles.

An implicit pole-assignment self-tuning regulator

A new self-tuning implicit pole-assignment algorithm is presented which, through the use of a pole compression factor and different RLS model and control structures, overcomes stability and convergence problems encountered in previously available algorithms. Computational requirements of the technique are much reduced when compared to explicit pole-assignment schemes, whereas the inherent robustness of the strategy is retained.

Auto-weighted minimum-variance self-tuning controller

A self-tuning controller which automatically assigns weightings to control and set-point following is introduced. This discrete-time single-input single-output controller is based on a generalized minimum-variance control strategy. The automatic on-line selection of weightings is very convenient, especially when the system parameters are unknown or slowly varying with respect to time, which is generally considered to be the type of systems for which self-tuning control is useful. This feature also enables the controller to overcome difficulties with non-minimum phase systems.

New developments in batch process input profile optimisation

A novel algorithm for solving nonlinear discrete time optimal control problems with model-reality differences is presented. The technique uses dynamic integrated system optimisation and parameter estimation (DISOPE) which achieves the correct optimal solution in spite of deficiencies in the mathematical model employed in the optimisation procedure. A new method for approximating some Jacobian trajectories required by the algorithm is introduced. It is shown that the iterative procedure associated with the algorithm naturally suits applications to batch chemical processes.

A genetic algorithm for the design of a fuzzy controller for active queue management

Active queue management (AQM) policies are those policies of router queue management that allow for the detection of network congestion, the notification of such occurrences to the hosts on the network borders, and the adoption of a suitable control policy. This paper proposes the adoption of a fuzzy proportional integral (FPI) controller as an active queue manager for Internet routers. The analytical design of the proposed FPI controller is carried out in analogy with a proportional integral (PI) controller, which recently has been proposed for AQM. A genetic algorithm is proposed for tuning of the FPI controller parameters with respect to optimal disturbance rejection. In the paper the FPI controller design metodology is described and the results of the comparison with random early detection (RED), tail drop, and PI controller are presented.

Active control comparisons: the ideal trial design

This report describes the concept for a clinical trial that uses carbamazepine as the gold-standard active control for a study of newly diagnosed patients. The authors describe an endpoint including efficacy and tolerability, and a stopping rule that uses a series of interim analyses in order to reach a conclusion as efficiently as possible without sacrificing reliability.

Reach & grasp therapy: Design and control of a 9-DOF robotic neuro-rehabilitation system

This paper presents the Gentle/G integrated system for reach & grasp therapy retraining following brain injury. The design, control and integration of an experimental grasp assistance unit is described for use in robot assisted stroke rehabilitation. The grasp assist unit is intended to work with the hardware and software of the Gentle/S robot although the hardware could be adapted to other rehabilitation applications. When used with the Gentle/S robot a total of 6 active and 3 passive degrees of freedom are available to provide active, active assist or passive grasp retraining in combination with reaching movements in a reach-grasp-transfer-release sequence.

Design, implementation and testing of an intelligent knowledge-based system for the supervisory control of a hot rolling mill

This paper describes the design, implementation and testing of an intelligent knowledge-based supervisory control (IKBSC) system for a hot rolling mill process. A novel architecture is used to integrate an expert system with an existing supervisory control system and a new optimization methodology for scheduling the soaking pits in which the material is heated prior to rolling. The resulting IKBSC system was applied to an aluminium hot rolling mill process to improve the shape quality of low-gauge plate and to optimise the use of the soaking pits to reduce energy consumption. The results from the trials demonstrate the advantages to be gained from the IKBSC system that integrates knowledge contained within data, plant and human resources with existing model-based systems. (c) 2005 Elsevier Ltd. All rights reserved.

Optimal observers for ARMA models

A polynomial-based ARMA model, when posed in a state-space framework can be regarded in many different ways. In this paper two particular state-space forms of the ARMA model are considered, and although both are canonical in structure they differ in respect of the mode in which disturbances are fed into the state and output equations. For both forms a solution is found to the optimal discrete-time observer problem and algebraic connections between the two optimal observers are shown. The purpose of the paper is to highlight the fact that the optimal observer obtained from the first state-space form, commonly known as the innovations form, is not that employed in an optimal controller, in the minimum-output variance sense, whereas the optimal observer obtained from the second form is. Hence the second form is a much more appropriate state-space description to use for controller design, particularly when employed in self-tuning control schemes.

Neurofuzzy control using Kalman filtering state feedback with coloured noise for unknown non-linear processes

This paper presents a controller design scheme for a priori unknown non-linear dynamical processes that are identified via an operating point neurofuzzy system from process data. Based on a neurofuzzy design and model construction algorithm (NeuDec) for a non-linear dynamical process, a neurofuzzy state-space model of controllable form is initially constructed. The control scheme based on closed-loop pole assignment is then utilized to ensure the time invariance and linearization of the state equations so that the system stability can be guaranteed under some mild assumptions, even in the presence of modelling error. The proposed approach requires a known state vector for the application of pole assignment state feedback. For this purpose, a generalized Kalman filtering algorithm with coloured noise is developed on the basis of the neurofuzzy state-space model to obtain an optimal state vector estimation. The derived controller is applied in typical output tracking problems by minimizing the tracking error. Simulation examples are included to demonstrate the operation and effectiveness of the new approach.